The long term goal of this investigation is to gain an understanding of how intracellular Ca2+ is regulated in airway smooth muscle. As a continuing research project, this proposal will address questions regarding how muscarinic cholinergic stimulation in bovine tracheal smooth muscle lead s to alteration of membrane conductance and subsequent changes in cytosolic free Ca2+. In addition to a standard electrophysiological approach the study will utilize double-barrelled Ca2+-selective microelectrodes with tip diameter smaller that 0.1 mum and a recently developed, laser excitation fluorescence imaging microscope. Determination of intracellular free Ca2+ in smooth muscle is still in its infancy despite rapidly developing non- invasive methods, notably a family of fluorescent indicators, as well as the traditional aequorin technique. This may, in part, reflect some limitations inherent to each methods, including recently identified problems with the fura-2 method. Recording with Ca2+ electrodes can determine the exact level of free Ca2+ concentration in cytosol and provide quantitative information on exact changes during agonist stimulation. For fast Ca2+ transient, a fluorescence digital imaging will be used. We will identify the receptor subtypes and document the effects of stimulation of each subtype on membrane conductance and [Ca2+]i. To this end, muscarinic antagonists will be screened in ligand binding studies which will be performed in single tracheal cells as well as membrane vesicle preparations. The studies will utilize isolated cells to overcome complications inherent in intact tissues, such as the presence of neural elements, heterogeneous population of cell types, and the mechanical and electrical coupling between cells. The combined study of three specific areas: 1) the identification of muscarinic receptor subtypes, 2) current clamp techniques and, 3) the quantization of intracellular [Ca2+] changes, will be very effective. Given the importance of intracellular Mg2+ in regulating [Ca2+], we will also examine Mg2+ changes in a similar manner. From these investigations, there should emerge a basic understanding of the regulation of intracellular [Ca2+] in airway smooth muscle, which will undoubtedly bridge further steps in elucidating the hyperexcitability of some disease states such as asthma.